Molecular analysis of muscarinic receptors and regulation mechanism of dilation in the rat iris.

采用RT-PCR方法准定量测定大鼠虹膜中毒蕈碱乙酰胆碱受体m1、m2、m3、m4和m5亚型mrna的数量。与其他平滑肌组织一样，大鼠虹膜中m2和m3的mRNA表达量高于m1和m5。大鼠虹膜的特点是m4 mRNA表达量高。此外，采用RT-PCR方法克隆大鼠虹膜中m2、m3和m4亚型的编码cdna。大鼠虹膜m2亚型氨基酸序列与心脏m2亚型氨基酸序列有9个差异，但与基因组序列完全相同。为了研究副交感神经切除对大鼠虹膜平滑肌影响的机制，我们测定了去睫状神经节大鼠虹膜中毒蕈碱受体亚型mRNA表达水平。结果表明，副交感神经切除对mRNA水平无明显影响。研究还表明，静息状态下大鼠虹膜扩张肌细胞内Ca^<2+>浓度（[Ca^<2+>]_i）高于虹膜括约肌和许多其他没有固有张力的平滑肌。由于毒蕈碱受体刺激导致大鼠虹膜扩张器[Ca^<2+>]_i降低，毒蕈碱松弛似乎是由于[Ca^<2+>]_i降低所致。在去神经虹膜扩张器中，毒蕈碱受体刺激既没有引起[Ca^<2+>]_i的降低，也没有引起松弛。

The amount of mRNAs of muscarinic acetylcholine receptor subtypes, m1, m2, m3, m4 and m5, in the rat iris was determined quasi-quantitatively using RT-PCR methods. The mRNA expression levels of m2 and m3 were higher than those of m1 and m5 in the rat iris as in other smooth muscle tissues. It was characteristic in the rat iris that the m4 mRNA expression level was high. In addition, the cloning of cDNAs encoding m2, m3 and m4 subtypes in the rat iris was performed using RT-PCR methods. Amino acids sequence of m2 subtype in the rat iris included nine differences in comparison with that in the heart, whereas it was 100% identical to genomic one. To examine the mechanisms underlying the parasympathectomy-induced effects in the rat iris smooth muscles, the mRNA expression levels of muscarinic receptor subtypes were determined in iris from ciliaryganglionectomized rats. The results suggested that the parasympathectomy does not markedly affect the mRNA levels. It was also clarified that the intracellular Ca^<2+> concentration ([Ca^<2+>]_i) in the rat iris dilator under the resting conditions is higher than those in the iris sphincter and many other smooth muscles which do not have an inherent tone. Since the muscarinic receptor stimulation resulted in the decrease in [Ca^<2+>]_i in the rat iris dilator, the muscarinic relaxation appeared to be due to the decrease in [Ca^<2+>]_i. In denervated iris dilators, muscarinic receptor stimulation induced neither the decrease in [Ca^<2+>]_i nor the relaxation.